Nucleic acid hybridization tests for the detection of specific DNA and RNA sequences are now common in research and are becoming common in diagnostic applications. These hybridization tests typically involve the use of a labeled nucleic acid probe in such assay formats as dot blots, Southern blots, Northern blots, in situ hybridization, plaque hybridization and colony hybridization. A variety of labels have been used in these assays including, for example, radiolabels, chemiluminescent compounds, enzymes and fluorescent compounds.
Almost as diverse as the labels are the methods of attaching the label to the nucleic acid probe. Briefly, the attachment of labels to a nucleic acid probe can be accomplished by either direct or indirect methods. Direct labeling is the result of attaching a label to a nucleic acid probe via a covalent linkage, typically prior to formation of a duplex. Alternatively, the label can be incorporated noncovalently into a duplex via intercalation. For indirect labeling, a hapten is attached to the nucleic acid probe, and later detected using a labeled specific binding protein.
One example of an indirect labeling system is the biotin-streptavidin system described in Langer, et al., Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981), the disclosure of which is incorporated herein by reference. In this system, a biotin moiety is attached to a nucleic acid probe and detection is carried out using a labeled avidin or labeled streptavidin. Methods for the attachment of biotin to the 5-position of a pyrimidine (e.g., uridine), the 8-position of a purine (e.g., guanidine) and the 7-position of a deazapurine (e.g., 7-deazaguanidine) have been described in U.S. Pat. Nos. 4,711,955, 5,241,060, 5,328,824, 5,449,767 and 5,476,928, each of which is incorporated herein by reference. While the biotin system is characterized by high sensitivity, an endogenous ubiquitous vitamin, vitamin H, is used as the modification group. This results in extraneous background signals, especially with biological samples.
Another indirect method involves the use of the hapten digoxigenin (see, Kessler, et al., Biol. Chem. Hoppe-Seyler 371:917-965 (1990)). This system uses the digoxigenin and antibody fragments derived from sheep polyclonal antibodies against digoxigenin. This method is also characterized by subpicogram sensitivity, and circumvents the problem of extraneous background signal by using the cardenolide digoxigenin which occurs only in Digitalis plants. Nevertheless, digoxigenin is an expensive reagent.
What is needed in the art are new methods of indirect labeling of probe:nucleic acid hybrids which have broad applicability, do not suffer from extraneous background signals and which provide modified duplexes which can be rapidly purified by affinity methods. Surprisingly, the present invention provides such methods, as well as the monomers and modified nucleic acids which are employed therein.